1. Field of the Invention
This invention relates to the recovery and purification of naturally produced chymosin. In particular, this invention is directed to methods for the recovery and purification of chymosin from aqueous solutions containing chymosin which have been obtained from the extraction of natural chymosin sources.
2. State of the Art
Chymosin is a known enzyme which is particularly useful in the preparation of cheese. While natural sources of chymosin include calf stomachs, bovine stomachs, goat stomachs, porcine stomachs, etc., commercial chymosin has heretofore been primarily obtained from the fourth stomach of milk fed calves. This is a result of the fact that such calves produce greater amounts of chymosin as compared to pepsin whereas other chymosin sources generally contain greater amounts of pepsin as compared to chymosin and accordingly, recovery of chymosin from such other sources is more difficult and economically less efficient, i.e., there is less chymosin to recover.
However, due to the recent decrease in calf production, the heretofore preferred natural source of chymosin has declined which, in turn, has provided impetus to developing more efficient methods for the recovery and purification of naturally produced chymosin. Specifically, more efficient methods would result in the improved recovery and purification of chymosin from calf stomachs as well as permit the economical recovery and purification of chymosin from other natural chymosin sources.
A major stumbling block to developing such methodology has been the very high level of contaminants found in the chymosin solution obtained from natural chymosin sources. In addition to pepsin, the aqueous extract obtained from these natural chymosin sources contain other contaminants including, for example, other stomach enzymes and proteins. Such contaminants have complicated the development of efficient recovery and purification methodology.
While numerous methods are disclosed for isolating enzymes from aqueous solutions, such as fermentation beer, none of the references which Applicants are aware of disclose methods for recovering and purifying naturally produced chymosin, especially naturally produced chymosin intermixed with pepsin and other contaminants, which employ a liquid-liquid two phase system.
In this regard, U.S. Pat. No. 4,144,130 describes the use of (1) a mixture of a high molecular weight unsubstituted or substituted polyalcohol, polyether, polyvinylpyrrolidone or polysaccharide and an inorganic salt, (2) a mixture of at least two of the above high molecular weight polymers to recover intracellular enzymes from an aqueous solution into which they have been released from the cells. When a mixture of polyethylene glycol and an inorganic salt is used, the desired intracellular enzyme goes into the top polyethylene glycol layer while the cell debris and other fermentation products go into the lower salt-containing layer. This reference discloses that the partition coefficients for various enzymes recovered in the glycol layer was about 0.3 when a normal cell mass was treated, which could be increased to about 3 when frozen cells were mixed with water and disintegrated to release their enzymes.
Similarly, U.S. Pat. No. 4,728,613, discloses a process for the recovery of extracellularly produced enzymes, such as protease, amylase and microbial rennet, from whole fermentation beer by using an inorganic salt in combination with a polymer selected from the group consisting of polyethylene glycol, an amine derivative of polyethylene glycol, a carboxylate derivative of polyethylene glycol, polypropylene glycol, an amine derivative of polypropylene glycol, a carboxylate derivative of polypropylene glycol, poly(ethylene glycol) ester, polyethyleneimine, trimethylamino-polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone and mixtures thereof. The examples of this reference disclose achieving partition coefficients of up to about 80 for such extracellular enzymes.
Likewise, Kula et al., "Purification of Enzymes by Liquid-Liquid Extraction", describes numerous methods for the purification of enzymes by liquid-liquid extraction. Among numerous methods disclosed, Kula et al. disclose that the addition of a polyethylene glycol/inorganic salt mixture to an aqueous solution containing the enzyme will form a two phase system wherein the polyethylene glycol phase will contain the enzyme. Kula et al. further disclose at page 111 that the phase forming polymer (polyethylene glycol) can be removed from the enzyme by adsorption of the enzyme onto ion exchangers; washing away of the phase forming polymer; and the subsequent recovery of the enzyme.
On the other hand, U.S. Pat. No. 4,508,825 discloses that extracellular protease and amylase co-produced during the fermentation of a microorganism capable of producing them are separated by the addition of polyethylene glycol and a cationic epihalohydrin/polyamine copolymer or dextran polymer to the fermentation medium and allowing the polymers to phase separate to form a protease rich phase and an amylase rich phase.
Also, U.S. Pat. No. 4,591,563, discloses a process for the simultaneous purification and concentration of the dextran-sucrase enzyme from the culture medium on sucrose. In particular, the disclosed method involves the addition of a polyether such as polyethylene glycol so as to form two phases; the first a heavy dextran-rich phase that contains the concentrated and purified dextran-sucrase enzyme, and the second a lighter polyether-rich phase that contains contaminating enzymatic activities, which is eliminate.
In view of the above, it is apparent that the cited art does not disclose the recovery and purification of naturally produced chymosin from aqueous solutions containing pepsin and other contaminants by employing an aqueous two phase system derived from the addition of polyethylene glycol and inorganic salt coupled with the use of an ion exchange resin. On the other hand, industrial or commercial scale recovery and purification of naturally produced chymosin is greatly facilitated by using such a liquid-liquid two phase system for the recovery of chymosin and by using an ion exchange resin for the purification of chymosin.
Accordingly, it is an object of this invention to provide efficient processes for the recovery and purification of naturally produced chymosin from aqueous mixtures containing chymosin, pepsin and other contaminants produced by the aqueous extraction of natural chymosin sources.
It is a further object of this invention to provide a recovery and purification process for naturally produced chymosin wherein recovery is achieved by using a liquid-liquid two phase system and purification is achieved by using an ion exchange resin.
It is still a further object of this invention to provide a recovery and purification process for naturally produced chymosin wherein recovery is achieved by using a liquid-liquid two phase system which provides for the selective recovery of chymosin and pepsin from other contaminants found in the aqueous solution.
It is a further object of this invention to provide a purification process for naturally occurring chymosin from an aqueous/polythylene glycol solution containing chymosin and pepsin.
These and other objects are achieved by the present invention as evidenced by the attached summary of the invention, detailed description of the invention, examples, and claims.